Vol.:(0123456789) Applied Microbiology and Biotechnology (2024) 108:191 https://doi.org/10.1007/s00253-023-12940-3 MINI-REVIEW Cleavage of natural rubber by rubber oxygenases in Gram‑negative bacteria Tulika Prakash1  · Sandhya R. Yadav1 · Marius Bürger2 · Dieter Jendrossek2 Received: 19 August 2023 / Revised: 6 December 2023 / Accepted: 14 December 2023 © The Author(s) 2024 Abstract Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Auran- tibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. Key points • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus Keywords Natural rubber · Poly(cis-1,4-isoprene) · Rubber oxygenase · RoxA · RoxB · RoxC · Haem · Dioxygenase · NR-degrading consortia · Aurantibaculum cummioxidans 35Y Introduction Natural rubber (NR) is present in over 2000 plant species including the rubber tree (Hevea brasiliensis). The latter produces a milky liquid (latex) in special tissues, the latic- ifers, that consists of an aqueous emulsion with poly(cis- 1,4-isoprene) as a main component (25 to 35% [wt/wt]) besides minor amounts of proteins and other components (Rose and Steinbuchel 2005). NR is an excellent polymer for the synthesis of several goods such as surgical gloves, tyres, toys, and medical devices. During the COVID-19 pan- demic, there was a huge increase in the NR waste generated from medical products. Most of the NR waste management methods are based on employing physical or chemical treat- ments, which have several health and environmental hazards (Arias and van Dijk 2019). NR-degrading microbes (bacteria and fungi) act on the polyisoprene chains and utilize them Tulika Prakash is the first author. All correspondence concerning this manuscript for bioinformatics and taxonomic aspects may be addressed to Tulika Prakash (tulika@iitmandi.ac.in) and that for the biochemical aspects on Rox proteins may be addressed to Dieter Jendrossek (dieter.jendrossek@imb.uni-stuttgart.de). * Tulika Prakash tulika@iitmandi.ac.in 1 School of Biosciences and Bioengineering, Indian Institute of Technology (IIT), Mandi, HP 175005 , India 2 Institute of Microbiology, University Stuttgart, Stuttgart, Germany http://orcid.org/0000-0001-9270-8775 http://crossmark.crossref.org/dialog/?doi=10.1007/s00253-023-12940-3&domain=pdf Applied Microbiology and Biotechnology (2024) 108:191 191 Page 2 of 9 as a carbon source for their growth. For previous overviews on NR-biodegrading bacteria, see Jendrossek et al. (1997), Rose and Steinbuchel (2005), Warneke et al. (2007), Yik- mis and Steinbüchel (2012), Ali Shah et al. (2013), and Chengalroyen and Dabbs (2013). The average time for NR degradation under laboratory conditions by Gram-positive microbes is 6 to 12 weeks; however, the rubber oxygenase of Gram-negative bacterium, Steroidobacter cummioxidans strain 35Y (earlier known as Xanthomonas sp. strain 35Y (Tsuchii and Takeda 1990; Sharma et al. 2018)), was the first to show ≈60% NR weight loss in only 1 week in in vitro experiments. The complete NR metabolism path- way has been elucidated by a combination of experimen- tal investigations and in silico analysis of the genome of S. cummioxidans 35Y (Sharma et al. 2018) as well as in a Gram-positive NR-degrader, namely Nocardia nova SH22a (Luo et al. 2014). For bacterial NR degradation, extracellular cleavage of NR into a mixture of small oligoisoprenoids is the first step. In Gram-negative bacteria, this cleavage is carried out by rubber oxygenases, namely RoxA and RoxB, which were originally identified in S. cummioxidans 35Y for the first time (Braaz et al. 2004; Jendrossek and Birke 2019). RoxA performs exo-cleavage and RoxB performs endo-cleavage of the polyisoprene molecule. As a result, the products differ with RoxA leading to the formation of (C15) oligoisoprenoids as main cleavage product and with RoxB leading to C20 or larger oligoisoprenoids (Jendrossek and Birke 2019). The purpose of this review article is twofold: first to review the recent developments that took place with regard to conservation, evolutionary, and functional aspects of the different types of rubber oxygenases and second is to highlight the evolutionary aspects of the bacteria exhibiting rubber degradation potential and harbouring these rubber oxygenases. Rubber oxygenases RoxA‑type rubber oxygenases (RoxAs) In addition to the RoxA35Y, other, at least, partially charac- terized RoxAs are those of Haliangium ochraceum SMP-2 (RoxAHoc), Corallocoocus coralloides BO35 (RoxACco), Myxococcus fulvus HW1 (RoxAMfu) (Birke et al. 2013), and Rhizobacter gummiphilus NS2 (Kasai et al. 2017) (Birke et  al. 2017). RoxAs have two binding motifs (CxxCH) for covalent attachment of the iron-containing cofactor haem thereby classifying them as dihaem c-type cytochromes (Jendrossek and Reinhardt 2003). Determina- tion of the RoxA35Y structure revealed a core in RoxA35Y that includes the two haem cofactors and is structurally almost identical to cytochrome c peroxidases (Seidel et al. 2013). The C-terminal haem of RoxA35Y is sixfold coordinated (with a Fe3+ ion, 4 N-Fe bonds from haem and two axial N-Fe-bonds from conserved main-chain histi- dine residues) but the N-terminal haem in RoxAs has only one axial ligand (His195) leaving the 6th position open. In RoxA35Y and RoxANS21, a dioxygen molecule is bound to the 6th coordination site of the haem iron (Fe2+-O2 −) (Jendrossek and Birke 2019; Seidel et  al. 2013). The N-terminal haem and the space above the oxygenated iron builds up the active site that is surrounded by a series of hydrophobic residues (A251, I252, L254, I255, F301, A316, and F317). The dioxygen molecule is stabilized by Phe317 (S. cummioxidans) corresponding to Phe302 in R. gummiphilus RoxA (Seidel et al. 2013) (Birke et al. 2012). Spectroscopic analysis revealed that the N-terminal active site haem is present in a reduced state (Fe3+---O2 −/Fe2+- -- O2 equilibrium) while the C-terminal haem is present in the oxidized Fe3+ form (Schmitt et al. 2019, 2010). A dioxygenase cleavage mechanism has been experimentally shown only for RoxA35Y (Braaz et al. 2005) but is likely also true for other RoxAs. RoxB‑type rubber oxygenases (RoxBs) The genomes of most Gram-negative NR-degrading bacteria have a second, roxA-related gene, coding for a protein shar- ing 35–40% identity with RoxAs including an N-terminal signal peptide for extracellular proteins and two haem bind- ing motifs (Jendrossek and Birke 2019; Kasai et al. 2017; Sharma et al. 2020). Heterologous expression of these genes of S. cummioxidans 35Y roxB35Y) and R. gummiphilus NS21 (latA) (in a S. cummioxidans background) and biochemical characterization of the purified proteins revealed that they catalyzed the dioxygen-dependent cleavage of poly(1,4-cis- isoprene) and thus represent true rubber oxygenases which have been designated as RoxB35Y and RoxBNS21, respectively (Birke et al. 2017; Jendrossek and Birke 2019). The products of RoxBs-catalyzed cleavage (endo-type) of polyisoprene differ from those of RoxAs (exo-type) in that the products are larger subunits (C20- or higher oligoisoprenoids, C25-, C30-, C35-, etc.) (Röther et al. 2017). Interestingly, a coop- erative effect on the efficiency of cleavage was determined when RoxA and RoxB were present simultaneously in an in vitro polyisoprene latex cleavage assay (Birke et al. 2018). A similar cooperative effect was noticed when RoxA was combined in vitro with Lcp. The produced oligoisoprenoids with different number of isoprene units could be quantita- tively isolated via gel permeation chromatography (Röther et al. 2017) or could be produced in enzyme bioreactors (Andler et al. 2020). Polyisoprene-derived oligoisoprenoids with functional groups (aldehyde and keto groups) are inter- esting compounds and could be useful building blocks in chemosynthetic and biotechnological applications. Applied Microbiology and Biotechnology (2024) 108:191 Page 3 of 9 191 Proteins related to RoxAs and RoxBs (RoxCs) A comprehensive in silico analysis identified 34 and 53 sequences similar to RoxA and RoxB, respectively (Sharma et al. 2020). A phylogenetic analysis of these Rox homo- logues revealed three putative clusters, namely ‘RoxBI’, ‘RoxAII’, and ‘RoxAIII and RoxBIII’ (Sharma et al. 2020). The cluster ‘RoxBI’ was formed by RoxB orthologs and the ‘RoxAII’ cluster was formed by RoxA orthologs; however, the third cluster ‘RoxAIII and RoxBIII’ was a mixed clus- ter formed by both RoxA and RoxB sequences. This third cluster in the phylogenetic tree suggested the emergence of another putative rubber oxygenase (RoxC) in the gamma- proteobacteria. Although RoxCs revealed similarities to either RoxAs or RoxBs, they differed from those in several aspects (for details, see Sharma et al. (2020)). For example, the signature motifs and residues of RoxC are more similar to RoxB as it harboured the CXXCH motif of haem 1, which was different from that of the sequences of the RoxAII clus- ter (CSXCH). In addition, members of the putative RoxCs missed some of the strictly conserved residues of active rub- ber oxygenases such as Phe317 or Trp302 (Jendrossek and Birke 2019). In July 2023, the BLAST search to identify new rub- ber oxygenases was repeated (see Sharma et al. (2020) for methodology) and the number of identified Rox- related sequences was found to be 458. Briefly, online BLASTP was used with default parameters to search for orthologs of the RoxA (UniProt-ID: Q7X0P3) and RoxB (UniProt-ID: A0A1S6Q8F9) protein sequences of S. cum- mioxidans 35Y against the UniProt (https:// www. unipr ot. org/, version as of July 2023) and the NCBI database of non-redundant protein sequences (NR) (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi, version as of July 2023). Orthologs were assigned as sequences with an E-value cutoff of 10−4, a threshold of 30% amino acid sequence identity, and 60% coverage of both query and hit. A phy- logenetic tree including all 456 Rox orthologues was constructed (Fig. 1 and suppl. Fig. S1) and consisted of three major clusters very similar to that described earlier (Sharma et al. 2020). The first cluster consisted of 117 sequences and was similar to ‘RoxAII’ (RoxAs, red in Fig. 1 and suppl. Fig. S1), the second cluster harboured 98 sequences and was similar to ‘RoxBI’ (RoxBs, green in Fig. 1 and suppl. Fig. S1), and the third largest cluster included 243 sequences and was similar to ‘RoxAIII and RoxBIII’, representing the putative RoxC group (RoxCs, blue in Fig. 1 and suppl. Fig. S1). Interestingly, one of the newly identified RoxA-related proteins was from Sphingomonas kyeonggiensis, a species of the alpha- proteobacteria. The identified Rox protein of this species revealed > 70% amino acids identity to RoxA35Y including a putative signal peptide and a MauG motif as well as two haem binding sites and conserved Phe317 and Trp302 residues. Therefore, it is likely that members of the alpha- proteobacteria also have biochemically active RoxAs and will turn out to be able to cleave and to utilize polyiso- prene as a carbon source. Similarly, RoxCs are no longer found to be restricted to gamma-proteobacteria. We have identified RoxC orthologues in the members beta-pro- teobacteria and Myxococcia. Another remarkable result was the identification of the first Gram-positive bacte- rium (Dietzia sp.) harbouring a putative roxB gene (Fig. 1 and suppl. Fig. S1). This RoxB-like protein, while having two haem binding sites and other RoxB-signatures, has a predicted molecular mass of only ≈50 kDa and lacked a signal peptide sequence. Since the protein sequence started with a serine (instead of methionine) and the DNA sequence of this protein was from a shotgun sequencing project, it is possible that the (true) start of the gene is missing and the resulting ‘complete’ polypeptide will turn out to have the missing RoxB features as well. Another interesting feature of most RoxC sequences is the presence of a signal peptide cleavage site with a conserved cysteine directly after the predicted cleavage site. This is typical for lipoproteins in which the protein is anchored with a covalently bonded acyl-glycerol to the (outer) membrane. We, therefore, predict that RoxC proteins will turn out to be attached to the (outer) cell membrane. Furthermore, a substantial number of RoxC sequences revealed the presence of an N-terminal domain harbouring a (third) haem binding motif (CxxCH) in addition to the two haem binding motifs of the putative catalytic domain related to RoxAs and RoxBs. (Fig. 1 and suppl. Fig. S1). The different features of the RoxA, RoxB, and RoxC proteins are summarized in Table 1. Unfortu- nately, no biochemical data for any protein of this third branch was available until recently. The (recent) expres- sion of two selected roxC genes (from Oceanococcus atlanticus and Fontimonas thermophilus) and partial puri- fication of the gene products (RoxCOat, RoxAFth) surpris- ingly could not demonstrate any rubber-cleaving activity and no polyisoprene latex–dependent oxygen consumption for these proteins (unpublished results). Furthermore, the theoretical isoelectric points (IEPs) of RoxCs generally were strongly acidic (IEP [RoxCFth] 5.1; IEP [RoxCOat] 4.6) while the IEP of RoxA35Y was substantially higher (pH, 7.3). The biological function of RoxCs is therefore currently unknown. The indication for RoxCs as lipopro- teins might point to a function in an oxidation of unsatu- rated fatty acids present in the cell wall of Gram-negative bacteria. https://www.uniprot.org/ https://www.uniprot.org/ https://blast.ncbi.nlm.nih.gov/Blast.cgi https://blast.ncbi.nlm.nih.gov/Blast.cgi Applied Microbiology and Biotechnology (2024) 108:191 191 Page 4 of 9 Evolutionary aspects of NR‑degrading enzymes and taxonomic position of strain 35Y All previously identified RoxA and RoxB homologues belong to the beta-, gamma-, and delta- classes of the phy- lum Proteobacteria (Sharma et al. 2020). Our recent BLAST search indicates the existence of at least one RoxB in alpha- proteobacteria also and RoxC in beta-proteobacteria and Myxococcia, in addition to gamma-proteobacteria. Horizon- tal gene transfer (HGT) is a potential mode of propagation of genes in different taxonomic lineages. Both roxA and roxB genes showed a number of interclass routes of HGTs among the members of phylum Proteobacteria. The roxA and roxB Applied Microbiology and Biotechnology (2024) 108:191 Page 5 of 9 191 genes were found to originate in the delta-proteobacteria and have later propagated to species of the beta- and gamma- proteobacteria (Sharma et al. 2020). The previous findings suggest that RoxA-, RoxB-, and RoxC-like proteins are (at present) restricted to Gram-neg- ative bacteria (Jendrossek and Birke 2019; Sharma et al. 2020, 2018); however, the recent results show that Gram- positive microbes can also harbour roxB genes. The extracel- lular cleavage of NR in most other Gram-positive bacteria is carried out by the latex clearing protein (Lcp). The so far only example of a Gram-negative microbe with an lcp gene (in addition to rox genes) is Solimonas fluminis HR-BB (Birke and Jendrossek 2019; Sharma et al. 2018). Although a synergistic activity of RoxA (RoxA35Y) with respect to RoxB (RoxB35Y) and Lcp (from Gram-positive Streptomyces sp. K30) was found, however, NR degradation properties of S. fluminis HR-BB have not been demonstrated till now. Percentage similarity of Lcp with RoxA and RoxB is 12.1% and 11.6%, respectively, confirming that Rox proteins and Lcp proteins are not related and have evolved independently. A phylogenetic analysis of the microbes harbouring rub- ber oxygenases revealed that both RoxA35Y and RoxB35Y are remarkably distinct from the other respective orthologues (Sharma et al. 2020). A similar taxonomic analysis using 16S rRNA and roxA and roxB genes of bacteria whose roxA gene products were confirmed to harbour rubber oxygenase activity also reveals a distinct branching of S. cummioxidans 35Y from the other microbes (Fig. 2). Besides, the rubber oxygenase degrading activity of S. cummioxidans 35Y is known to be superior over other investigated NR-degrading species in its activity leading to a relatively fast NR degrada- tion. This raised significant interest among the researchers to explore the evolutionary and taxonomic aspects of this special microbe. Initial reports on S. cummioxidans 35Y termed it as Xanthomonas 35Y and considered it as a mem- ber of the order Xanthomonodales within gamma-proteo- bacteria (Tsuchii and Takeda 1990). In 2015, Naushad et al. reclassified Xanthomonodales and proposed a novel order Nevskiales under the class gamma-proteobacteria (Naushad et al. 2015). Subsequently, using a polyphasic taxonomic approach on several features including genomic, physiologi- cal, biochemical, and chemotaxonomic traits, three families in the order Nevskiales, namely, Algiphilaceae, Sinobacte- reaceae (Solimonodaceae), and Salinispharaceae, were pro- posed (Naushad et al. 2015; Sharma 2019); however, only the former two families were classified in the NCBI taxon- omy database at that time. With the availability of the whole genome sequence of S. cummioxidans 35Y in 2018 and the 16S rRNA gene sequences of additional Steroidobacter spe- cies, the tentative taxonomic positioning of genus Steroido- bacter was also revised (Sharma 2019) and it became obvi- ous that strain 35Y could not be a species of Xanthomonas but represents (at least) a new species of a new genus for which the designation Steroidobacter cummioxidans 35Y was chosen (Sharma et  al. 2018). An in silico analysis revealed that the genera Steroidobacter and Povalibacter, which are a part of Sinobactereaceae, clustered separately as a sub-clade from the rest of the members of the families Sinobactereaceae and Algiphilaceae. This led to the proposi- tion of a novel family Steroidobacteraceae fam. nov. within the order Nevskiales by Sharma (2019) to accommodate three genera, including Steroidobacter and Povalibacter. The proposal for a novel family Steroidobacteraceae fam. nov. was also made by Liu et al. (2019) based on 16S rRNA gene sequence analysis while annotating a novel microbe Stenotrophobium rhamnosiphilum (GT1R17T) isolated from a glacier. Recently, genetic-based analysis proposed the new order Steroidobacterales which includes the family Steroi- dobacteraceae (Montecillo 2023). Interestingly, the whole genome sequence–based analysis clearly separated S. cummioxidans 35Y from the other spe- cies of the genus Steroidobacter by its nearly double size of the genome (≈7.9 Mb for 35Y compared to 3.5 Mb for S. denitrificans). Although S. cummioxidans 35Y exhibits high 16S rRNA gene sequence identity with a few species of genus Steroidobacter that would point to be a member of the genus Steroidobacter, the analysis of the whole genome sequences ANI (average nucleotide identity) and in silico DDH (DNA-DNA hybridization) could not classify strain 35Y into any of these species (Sharma et al. 2018). This was further supported by POCP (percentage of conserved proteins), ANI, and 271 proteins based core AAI (average amino acid identity) analysis (Sharma 2019). In addition, the morphological and physiological features of S. cummiox- idans 35Y were found to be different from those of the type species of genus Steroidobacter (S. denitrificans) (Sharma Fig. 1 Phylogenetic relationships among orthologs of newly identi- fied rubber oxygenases (RoxA and RoxB). The phylogenetic tree was constructed based on protein sequences of the RoxA and RoxB orthologs by using the neighbor-joining method (Bootstrap 1000). The cytochrome c peroxidase protein of Geobacter sulfurreducens (DSM 12127) was used as the outgroup (coloured in brown). Solid lines represent the lengths of branches; dotted lines are used to align the tip labels for better visualization. From each of the three groups of the full phylogenetic tree (suppl. Fig.  S1), one species of every group was selected as representative. Red, green, and blue solid lines indicate enzymes which were assigned to the RoxA (22 sequences), RoxB (19 sequences), and RoxC (19 sequences) groups, respectively. Enzymes with a red and green background were expressed and bio- chemically characterized as RoxAs and RoxBs previously; however, enzymes with a blue background were expressed and biochemically characterized but showed no rubber oxygenase activity (unpublished data). Enzymes which contain a third N-terminal haem are indicated by a ‘3.haem’ suffix at the end of their name. Two bacteria of new classes were identified to contain ‘Rox-like’ enzymes. The alpha- proteobacteria Sphingomonas kyeonggiensis (indicated by *) and the GC-rich Gram-positive bacterium Dietzia sp. (indicated by **) are the first examples of rubber oxygenase orthologs found outside of the beta-, gamma-, and delta-proteobacteria classes ◂ Applied Microbiology and Biotechnology (2024) 108:191 191 Page 6 of 9 2019; Fahrbach et al. 2008). Significant differences of S. cummioxidans 35Y to the other species of genus Steroido- bacter were also identified with respect to the utilization of carbon sources and growth conditions (D. Jendrossek, unpublished data; Sharma 2019)). The chemotaxonomic fea- tures of S. cummioxidans 35Y were significantly different from the other members of genus Steroidobacter (Sharma et al. 2018). These important differences in the physiologi- cal, biochemical, and chemotaxonomic and genome-charac- teristics between S. cummioxidans 35Y and the type species of the genus Steroidobacter suggest that S. cummioxidans 35Y is a species of a novel genus. Based on these differ- ences, S. cummioxidans 35Y was suggested to represent the first species of a new genus for which the designation Auran- tibaculum gen. nov. had been proposed (D. Jendrossek, unpublished data; Sharma 2019). The taxonomic designation of strain 35Y would be Aurantibaculum cummioxidans 35Y (golden rod utilizing rubber). Microbial consortia–based NR biodegradation Recently, the use of microbial consortia for NR biodegrada- tion has been attempted by adopting different strategies of using mono-cultures, mixed cultures, natural consortia, syn- thetic microcosms, or consortia formulated by using enrich- ment techniques (Veenagayathri and Ahongsangbam 2017; Nawong et al. 2018; Nguyen et al. 2020; Bosco and Mollea 2021; Cheng et al. 2024). It is important to note that a direct comparison of NR biodegradation of individual species and consortia is necessary in the future as the results of the Table 1 Properties of rubber oxygenases RoxA35Y, RoxA from S. cummioxidans 35Y; RoxB35Y, RoxB from S. cummioxidans 35Y; RoxCFth, RoxC from Fontimonas thermophila; RoxCOat, RoxC from Oceanococcus atlanticus; ODTD, 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al; -: feature not present; pattern: mixture of C20, C25, C30,…..and higher oligoisoprenoids Protein/attribute RoxA35Y RoxB35Y RoxCFth RoxCOat Gene length [bp] 2037 2046 2454 2136 Secretion system Sec Sec Sec (lipoprotein) Sec (lipoprotein) Signal peptide cleavage site ALA/L17TP TDA/A34TD LGG/C21AG LVA/C16NG Mw pre-protein [kDa] 74.7 73.8 88.9 77 Mw mature protein [kDa] 71.5 70.3 86.9 75.4 IEP mature (theor.) 7.3 6.3 5.04 4.63 % of arom. AA 11.4 9.9 9 8.9 Total no.: F, Y, W (24, 30, 20) (23, 24, 17) (31, 24, 17) (26, 20, 16) Available structures X-ray (1.80 Å) Alphafold Alphafold Alphafold Haem attachment N-terminal CSACH195 CHACH196 CFQCH312 CFQCH204 C-terminal CASCH394 CASCH395 CASCH538 CASCH437 3. haem (N-terminal) no no CRTCH44 no Axial haem ligands N-terminal H195 H196 H312 H204 C-terminal H394 H641 H395 H627 H538 H776 H437 H675 3. haem (N-terminal) - - H 44 H 98 - Fe state ‘as isolated’ Fe2+–O2/Fe3+ Fe3+/Fe3+ Fe3+/Fe3+/Fe3+ Fe3+/Fe3+ MauG motif PYFH517NGSVP PYMH494NGSVP PYDH673NGSVP PYLH553 NGSVP F317 equivalent (according to structural alignment) F317 F309 A474 A354 W302 equivalent (according to structural alignment) W302 V294 F459 F339 Soret max (ox) [nm] 407 404 409 408 Soret max (reduced) [nm] 418 419 418 418 Beta (reduced) [nm] 549, 553 548, 556 551 551 Alpha (reduced) [nm] 521 522, 529 522 522 Bands above 600 nm No ~ 618 No No UV/Vis effect upon addition of CO Yes No No - Cleavage product(s) ODTD Pattern No rubber oxygenase activity No rubber oxygenase activity Applied Microbiology and Biotechnology (2024) 108:191 Page 7 of 9 191 consortia cannot be directly compared with those obtained using pure cultures. In principle it is, however, possible that the use of consortia may be more beneficial than pure cul- tures in the biodegradation of natural rubber. The use of consortia might also be beneficial to degrade chemically modified (e.g. vulcanized) rubber. Summary The biodegradation of NR in Gram-negative proteobacteria is carried out by two types of rubber oxygenases, namely RoxA and RoxB. These enzymes differ in their catalytic mode of action. However, both these enzymes work in a synergistic manner in the case of S. cummioxidans 35Y. A third type of Rox-related proteins (RoxCs) is also pre- sent in many Gram-negative microbes, however, at present restricted to species of the beta- and gamma-proteobacte- ria. The rubber oxygenases of S. cummioxidans 35Y appear to be superior thereby leading to a relatively fast NR bio- degradation process. The phylogenetic analysis using the whole genome sequence of S. cummioxidans 35Y led to the proposal of three families in the order Nevskiales including the novel family Steroidobacteraceae fam. nov. Within the genus Steroidobacter, the physiological properties and the doubled genome size of S. cummioxidans 35Y compared with the other species of genus Steroidobacter suggest that strain 35Y is a species of a novel genus (Aurantibaculum gen. nov.) within the Steroidobacteraceae. Supplementary Information The online version contains supplemen- tary material available at https:// doi. org/ 10. 1007/ s00253- 023- 12940-3. Acknowledgements This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to DJ. MB and DJ acknowl- edge technical support by Chiara Brueck during biochemical char- acterization of RoxC. This study was supported by the Science and Engineering Research Board (SERB), Government of India to TP with grant number CRG/2019/006295. Author contribution TP conceived and designed the idea. TP, SY, and MB contributed to the bioinformatics and taxonomic aspects. MB and DJ contributed to the biochemical aspects on the Rox proteins. TP, SY, and DJ wrote the manuscript. All authors read and approved the manuscript. Fig. 2 Phylogenetic relationships between S. cummioxidans 35Y and the other bacteria, whose RoxA proteins have been found to harbour rubber oxygenase activity, on the basis of A RoxA protein, and B RoxB protein, and C 16S rRNA gene sequences. For insets A and B, the phylogenetic tree was constructed based on amino acid sequences of the RoxA and RoxB orthologs by using the neighbor- joining method. The cytochrome c peroxidase amino acid sequence of Thermosulfurimonas dismutans was used as the outgroup. For inset C, the phylogenetic relationships between S. cummioxidans 35Y was inferred by using the Maximum Likelihood method and Kimura 2-parameter model  (Kimura 1980). Initial tree for the heu- ristic search was obtained automatically by applying Neighbor-Join (Saitou and Nei 1987)  and BioNJ algorithms to a matrix of pair- wise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. Bootstrap value (Felsenstein 1985) (resampled 1000 times) is shown in % and values less than 50% are not shown. Evolu- tionary analyses was performed in MEGA X (Kumar et al. 2018) and the phylogenetic tree was formatted using figtree https://doi.org/10.1007/s00253-023-12940-3 Applied Microbiology and Biotechnology (2024) 108:191 191 Page 8 of 9 Data availability All data supporting the findings of this study are available within the paper and its Supplementary Information. Declarations Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Ali Shah A, Hasan F, Shah Z, Kanwal N, Zeb S (2013) Biodegradation of natural and synthetic rubbers: a review. 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